The complete information for the chain folding which is present in the functionally active state of a protein is contained in the amino acid sequence of the protein. This statement has been proved experimentally by the fact that correct refolding of artificially unfolded proteins has been demonstrated in vitro, even if only in selected cases (Anfinsen, C.B., Science 181: 223-230, 1973). However, there are very many gaps in the understanding of the process of protein folding, in particular of complex proteins with multiple domains or several subunits, (Janicke, R., Biophys. Struct. Mech. 8: 231-256, 1982) and no substantial progress has been achieved in this field in recent years. The study of protein folding in vivo has been neglected completely since it has been presumed that, in principle, proteins fold in a spontaneous self assembly process even under physiological conditions (Creighton, T., in "Proteins", Freeman and Company, New York, 1984). However, this notion is most probably wrong. Many proteins which attain their functional conformation in from seconds to a few minutes after synthesis in vivo cannot be refolded in vitro after unfolding under various denaturing conditions. Recently there have been more and more findings which have led to the hypothesis that the conformation of protein chains is physiologically influenced by specific components, essentially heat shock proteins (Pelham, H., Nature 332: 776-777, 1988). Heat shock proteins are proteins which are present in the cells of organisms and are formed to a larger extent when the cells are subjected to particular forms of stress such as, for example, heat stress.
One protein component which catalyzes the correct chain folding of unfolded or newly synthesized polypeptides in vivo and in vitro has already been identified and functionally characterized This component is the heat shock protein GroEL or Hsp60 which occurs in bacteria and mitochondria. There is 60% identity of the amino acid sequences of GroEL in Escherichia coli and Hsp60 in mitochondria (Hemmingsen, S.M. Woolford, C., van der Vies, S.M. Tilly, K., Dennis, D.T., Georgopoulos, C.P. Hendrix, R.W. and Ellis, R.J., Nature 333: 330-334, 1988; Reading, D.S. Hallberg, R.L., Myers, A.M., Nature 337: 655-659, 1989). The components are present as virtually identical oligomeric complexes of 14 subunits with molecular weights of about 58,000 dalton each. Artificially unfolded or nascent proteins bind to the surface of the GroEL or Hsp60 complex. There is no specificity for bacterial or mitochondrial proteins in this case. The chain folding takes place on GroEL or Hsp60 in a reaction dependent on the hydrolysis of ATP (=adenosine triphosphate). The involvement in this process of a further component associated with the GroEL or Hsp60 complex cannot be ruled out. In the absence of ATP, for example the murine enzyme dihydrofolate reductase binds to Hsp60 in the form of an unfolded highly protease-sensitive polypeptide chain. ATP addition leads to a substantial increase in dihydrofolate reductase activity, which is initially still associated on the surface of Hsp60, and there is subsequent release of the enzyme which then is in its enzymatically active conformation. This reaction can take place inside the mitochondria at a protein concentration of about 30 to 50 g of protein/100 ml. However, it is in principle possible with isolated Hsp60 in vitro in the same way. Elimination of the Hsp60 function in a conditional yeast mutant leads to failure of mitochondrial proteins to assemble to supramolecular complexes (Cheng, M.Y., Hartl, F.U., Martin, J., Pollock, R.A., Kalousek, F., Neupert, W., Hallberg, E.M., Hallberg, R.L., and Horwich, A.L., Nature 337: 620-625, 1989).
GroEL and Hsp60 are soluble, relatively insensitive to nonspecific proteases such as proteinase K and can easily be isolated in substantial amounts from E. coli and the like. A method of isolation for GroEL is described, for example, in the reference Hendrix, R.W., J. Mol. Biol. 129: 375-392 (1979) GroEL is named gp groE therein. The protein gp groE is synonymous with the protein GroEL.
In the case of the isolation of Hsp60 from mitochondria, an extract which has been prepared in 0.3 % digitonin, 100 mM NaCl, 30 mM tris (=tris(hydroxymethyl)aminomethane) at pH 7.4 provides the protein Hsp60 in a good yield and sufficient purity in two purification steps by ammonium sulfate precipitation (35 to 50% saturation) and Q-.RTM.Sepharose chromatography; Q-Sepharose is a basic ion exchanger based on agarose from Pharmacia LKB GmbH, Freiburg (Federal Republic of Germany).